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J Bacteriol. Jun 2000; 182(12): 3553–3558.
PMCID: PMC101956

A Common Step for Changing Cell Shape in Fruiting Body and Starvation-Independent Sporulation of Myxococcus xanthus

Abstract

Myxococcus xanthus can sporulate in either of two ways: at the end of the program of fruiting body development or after exposure of growing cells to certain reagents such as concentrated glycerol. Fruiting body sporulation requires starvation, while glycerol sporulation requires rapid growth, and since the two types of spores are structurally somewhat different, it has generally been assumed that the two processes are different. However, a Tn5 Lac insertion mutation, Ω7536, has been isolated which simultaneously blocks the development of fruiting body spores as well as glycerol-induced spores. Both sporulation pathways are blocked in the mutant within the process that converts a rod-shaped cell into a spherical spore. The Ω7536 locus is expressed at the time of cell shape change appropriate to each process, early after glycerol induction and late after starvation induction. On the C-signal response pathway, it is possible to identify positions for the normal function of the Ω7536 locus and for the inducing stimulus from glycerol that are unique and consistent with the observations. Although the two sporulation pathways differ in certain respects, it is shown that they share at least one step for changing a rod-shaped cell into a spherical spore.

In response to nutrient limitation, Myxococcus xanthus cells assemble a multicellular fruiting body within which myxospores are produced (3, 8, 23). Individual, rod-shaped prespores differentiate into the spherical myxospores, which are metabolically quiescent and environmentally resistant. Sporulation and aggregation are responses to the same extracellular signal, the approximately 20-kDa C-factor protein. This signal, the product of the Myxococcus csgA gene, is associated with the cell surface (10, 12, 24). Mutant csgA cells are defective in aggregation and fail to sporulate. However, if they are either mixed with wild-type cells or supplemented with purified C-factor, their abilities to aggregate and sporulate are restored (10, 24). C-factor is produced in response to starvation (10).

There is a second way for Myxococcus to sporulate, called starvation-independent sporulation. When cells are growing vigorously in a rich, aerated medium, exposure to certain reagents, such as 0.5 M glycerol (4), 0.7 M dimethyl sulfoxide (DMSO) (13), phenyl ethanol (4), or a variety of β-lactam antibiotics (22), induces single cells to become spores within 2 to 3 h. From cell to cell, glycerol-induced shape changes are quite synchronous, and almost all treated cells become spores. This type of sporulation is promoted by ample nutrient and vigorous aeration, not starvation. The nature of starvation-independent spores is the same whether the inducer is glycerol or DMSO, suggesting that these reagents induce an endogenous process of sporulation, which may be otherwise induced and augmented in the process of fruiting body development.

Fruiting body sporulation, also known as starvation-dependent sporulation, is cell cooperative, depends on cell density, requires a surface like agar, and occurs subsequent to aggregation. Often no more than 1% of input cells become viable fruiting body spores, and it takes at least 3 days to complete spore maturation. In addition, there are structural differences between the two types of spores. The starvation-independent spores lack the fruiting body spore protein S (13), but they contain many more ribosomes, and their coats are thinner (28). On the one hand, many developmental mutants that cannot form mature fruiting bodies can still form viable glycerol spores, including asgA, asgB, asgC, csgA, and dev mutants (6, 19). On the other hand, many mutants that have lost glycerol inducibility still form spores in fruiting bodies (1).

Despite their differences, the starvation-dependent and starvation-independent pathways may have steps in common, steps that change the cell shape from a rod into a sphere, add thickness to the wall, and enhance resistance to injury. Both types of spores contain protein U (13); both pathways induce a β-lactamase activity (22).

Here, we report a mutation that causes defects in the change in cell shape. This mutation affects fruiting body and starvation-independent sporulation in similar ways and identifies a particular step common to both.

MATERIALS AND METHODS

Bacterial strains, media, growth, and development.

The M. xanthus strains used in this study were as follows. DK 1622 (wild type) (9) and DK 5208 (Tn5-132::csgA) (15) have been described elsewhere. The origin and properties of DK 7536 (Tn5lac Ω7536) are presented in the text. New genotypes were constructed using the transducing phage Mx4ts18ts27 hrm (2) or Mx8clp2 (21). DK10524 (Tn5lacΩ7536) was produced by a transductional backcross of DK 7536 into DK 1622. DK10527 (Tn5lacΩ7536 Tn5-132::csgA) was constructed by transduction from DK10524 into DK5208 with kanamycin selection. DK10552 (Tn5lac Ω7536 ΔdevRS) was constructed by transduction with kanamycin selection from DK10524 into the dev mutant DK11212, produced by Bryan Julien of Kosan Biosciences.

TPM buffer is 10 mM Tris-HCl (pH 8.0)–8 mM MgSO4–1 mM KPO4 (pH 7.6). Myxobacteria were grown at 32°C in CTT broth (1% Bacto Casitone in TPM buffer) or CTT agar (CTT broth solidified with 1.5% Bacto Agar). CTT soft agar contained 0.7% Bacto Agar. Kanamycin (40 μg/ml) or oxytetracycline (12.5 μg/ml) was added when indicated. Growth was monitored with a Klett-Summerson photoelectric colorimeter equipped with a red filter. To monitor developmental aggregation, cell suspensions containing 5 × 109 cells/ml in TPM buffer were deposited onto TPM agar plates (TPM buffer plus 1.5% Bacto-Agar) as described elsewhere (7). Starvation-induced sporulation was measured in submerged culture as described previously (17). After 3 or 5 days of incubation at 32°C, the samples in each well were sonicated for 10 s at 40% output using a standard microtip. Samples were transferred from the microtiter wells to microcentrifuge tubes and sonicated for an additional 5 min in a cup horn with ice water cooling. Following sonication, the samples were heated for 2 h at 50°C.

For induction of starvation-independent sporulation, glycerol was added to exponentially growing M. xanthus cultures in CTT medium to a final concentration of 0.5 M and incubated with shaking at 32°C. A sample of the culture was diluted and plated immediately to determine the initial number of viable cells. In experiments to determine the resistance properties of cells produced after glycerol exposure, the samples were heated to 33 or 40°C. To determine resistance to detergent, sodium dodecyl sulfate (SDS) was added to the cell suspensions to a concentration of 0.01% for 30 min at room temperature. After serial dilution of the samples, aliquots were plated on CTT agar with or without antibiotics and incubated at 32°C. Three to five days later, spore titers were calculated based on the number of visible colonies.

To determine the abilities of strains either to rescue other developmentally defective strains in spore formation or to be rescued from their own sporulation defect, the strains in question were mixed in a 1:1 ratio before being deposited into microtiter wells for submerged culture. Strains could be differentiated by selecting for the appropriate antibiotic marker—either kanamycin (40 μg/ml) or oxytetracycline (12.5 μg/ml).

Tn5lac mutagenesis and isolation of developmental mutants.

Tn5lac was introduced into the fully motile strain DK1622 by P1 transduction (14). Kmr transductants were picked to fresh CTT-plus-kanamycin plates. Approximately 10,000 transductants were examined for increased lacZ expression during development over a 3-day period by Harvey Kimsey. Vegetatively growing cells were transferred on toothpicks from CTT agar to TPM agar plates containing 40 μg of X-Gal (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside)/ml or CTT agar plates containing 20 μg of X-Gal/ml. Levels of β-galactosidase activity were estimated by comparing the intensity of blue dye deposited by developing cells versus vegetatively growing cells. In all, 272 strains were retained because they displayed increased lacZ expression during development.

To identify insertion mutations that disrupted M. xanthus development, the Kimsey set of 272 Tn5lac insertion strains was then screened for the capacity to form fruiting bodies and spores. Vegetatively growing colonies of each strain were transferred on toothpicks to TPM agar and allowed to develop at 33°C for 3 or 5 days. At the end of the incubation period, the stabs were heated for 2 h at 50°C to kill the vegetatively growing cells and overlaid with soft agar that contained CTT with a higher concentration of Casitone such that the final concentration of Casitone on the plate was 1%. Three days later the stabs were scored for germination and growth. Spores survive the 50°C treatment and are able to germinate when overlaid with CTT, but mutations that block development either prevent the formation of heat-resistant spores or greatly reduce their number. Consequently, there is no growth, or a smaller colony, after the 1% Casitone overlay. The amount of growth for each strain was compared with those for DK1622, the wild-type strain, and DK5208, a csgA mutant strain, which were stabbed to each screening plate. Though not quantitative, the stabs do reveal strains that are severely defective in myxospore formation. The stabs are convenient for screening many strains. Cultures of possible mutant strains were checked quantitatively. To determine their capacities to swarm and and to develop fruiting body-like aggregates on TPM agar, mutant strains were examined microscopically.

Measurement of β-galactosidase activity from Tn5lac.

Cells were induced to develop either in submerged culture or on TPM plates. If they were developed on plates, 100 μl of a cell suspension of 5 × 109 cells/ml was spotted in 20-μl aliquots. At intervals, cells were harvested from these plates into 400 μl of TPM buffer and stored at −20°C until all samples were collected. If cells were developed in microtiter wells, entire dishes were removed from the 32°C incubator and stored at −20°C until all samples were collected. Samples obtained by either method were treated as follows (adapted from reference 18). The samples were dispersed by a 10-s exposure to a microtip sonicator from Heat Systems-Ultrasonics. Samples from microtiter wells were transferred to microcentrifuge tubes. The samples, now all in tubes, then underwent a second sonication for 5 min in a cup horn (Tekmar) with ice water cooling. Before this second sonication, glass beads (acid washed; diameter, 425 to 600 μm) were added to the sample tubes to disrupt spores, which otherwise resist shear breakage by sonication alone. β-Galactosidase specific activity was assayed as described by Kuspa et al. (18), with two modifications: β-mercaptoethanol was omitted from the Z buffer, and samples were not sedimented before they were assayed. To assay β-galactosidase from cells induced to sporulate under nonstarvation conditions, 0.5 M glycerol was added to the cultures at time zero. At each time point, 0.5 ml was withdrawn, the suspension was centrifuged, and the supernatant discarded. The pellet was stored at −20°C. When all samples had been collected, the cell pellets were thawed and resuspended in 1 ml of TPM buffer. Glass beads were added to each sample, and samples were sonicated in a cup horn sonicator and immediately assayed for β-galactosidase activity.

Scanning electron microscopy.

To reveal the cellular structures inside a fruiting body, cells were allowed to develop on Nuclepore polycarbonate filters. Cell suspensions (20-μl droplets of 5 × 109 cells/ml in TPM buffer) were placed onto the filters, and the filters were placed onto a TPM agar plate. The droplets of cells were allowed to dry into the filters before the plates were inverted to incubate at 32°C for 3 days. After development, the fruiting body-like aggregates were fixed by immersing the filters in a 2.5% glutaraldehyde-veronal acetate solution overnight. Following glutaraldehyde fixation, the filters were transferred into a 1% OsO4-veronal acetate solution for 1 h. Specimens were then stained for 30 min with 1% uranyl acetate before dehydration in a graded series of ethanol solutions (15, 30, 45, 60, 75, and 90%) for at least 1 h each. Finally the specimens were incubated in 100% ethanol for at least 1 h, and they were chemically dryed with hexamethyldisilane. The fruiting bodies were then dry fractured with a razor blade. The fractured samples were gold coated to a thickness of 11 nm, using a Polaron SEM coating system. Specimens were examined with a Philips 505 scanning electron microscope.

RESULTS

Identification of sporulation mutant Ω7536.

A collection of 272 strains with independent Tn5lac insertions in M. xanthus, having developmental defects and developmentally regulated expression of β-galactosidase (described in Materials and Methods), was screened for mutants that were defective specifically in sporulation. Identified in this screen, insertion mutant Ω7536 produced fewer than 10−6 as many heat- and sonication-resistant spores as the wild type, measured by the capacity to form colonies after such treatment. Actually, 2 × 108 cells were plated on each of several plates, and there were no colonies on any plate. Despite this extreme sporulation defect, mutant Ω7536 aggregated well (Fig. (Fig.1).1). Its aggregates are similar in number, size, and shape to those of the wild type.

FIG. 1
Aggregate formation. Photographs were taken at 48 h of development on TPM agar. (A) The wild-type strain DK1622; (B) DK 7536. Bar, 0.2 mm.

Fruiting body cell morphology.

The screen utilized to isolate the Ω7536 insertion mutant would not have distinguished between a defect in sporulation and one in germination. To address this issue, the cells within wild-type and Ω7536 fruiting bodies were examined by scanning electron microscopy, which, unlike a colony assay, does not rely on germination (Fig. (Fig.2).2). After 3 days of development, the wild type forms spores that pack very regularly within a fruiting body because of their uniform size, spherical shape, and high density. Within the Ω7536 mutant fruiting body-like aggregates, however, a heterogeneous mixture of cell sizes and shapes is seen. The shapes range from shortened rods to bean shapes to ovoids of various proportions. Because these shapes could be intermediates in the morphogenesis of spores, their accumulation would imply that Ω7536 has a defect in spore morphogenesis. Also, because of their variety of shapes, the cells within an Ω7536 fruiting body are not packed in an orderly fashion, in striking contrast to the organization of cells in a wild-type fruiting body (Fig. (Fig.2).2).

FIG. 2
Scanning electron micrographs of aggregates and their contents. Photographs were taken at 48 h of development on TPM agar. (a and b) The wild-type strain, DK1622, at low and high magnifications, respectively. (c and d) DK 7536 at low and high magnifications, ...

Even though the insertion mutation profoundly affects the cell's ability to change its shape and to become heat and sonication resistant, there are no obvious delays in forming the fruiting body aggregates. Nor does the mutation disrupt or impair growth at 32°C in CTT medium (data not shown). Swarming defects often impair or delay aggregation (8, 15). But the mutant has no motility defect revealed by its swarming pattern (data not shown). That pattern is produced by the functions of two nonoverlapping sets of multiple genes, known as A and S (9). Crosses that would separate the A and S motility systems from each other to reveal either an A or an S defect disclosed neither in the Ω7536 mutant, which therefore is genetically A+ S+.

Gene expression from the Ω7536 insertion.

Since the mutation arises from an insertion of the Tn5lac transposable element (14), which is properly oriented to fuse the affected locus transcriptionally to lacZ, expression of the locus can be monitored by measuring β-galactosidase specific activity (Fig. (Fig.3).3). The Ω7536 insertion is not expressed vegetatively; it is induced after 17 h of development in submerged culture, a time that matches the normal beginning of sporulation after the onset of starvation. Moreover, in the wild type, sporulation is completely dependent on the extracellular C-signal (20, 26), so that csgA mutants form fewer than 10−6 spores of the signal-competent strain. To test the C-signal dependence of the Ω7536 gene, we transduced the insertion into a strain that cannot make C-signal. The csgA-deficient mutant of Ω7536 does not express its β-galactosidase (Fig. (Fig.3).3).

FIG. 3
Expression of β-galactosidase, during development, from the Tn5lac promoter fusion Ω7536. Cells were sampled immediately after transfer to starvation buffer for the 0-h point. Expression was measured in a wild-type (DK1622) background ...

This combination of mutant morphology and gene expression strongly suggests that the gene identified by the Ω7536 insertion is directly involved in spore formation. In light of the virtually normal aggregation by the mutant, it appears that its defect is specific to sporulation. If that gene is involved in the change in shape from a rod to a sphere, then the Ω7536 insertion might alter starvation-independent sporulation, which is induced by 0.5 M glycerol, phenyl ethanol, β-lactam antibiotics, and other agents (4, 13, 22).

Starvation-independent sporulation of Ω7536.

If wild-type M. xanthus cells are exposed in a rich, well-aerated growth medium to any one of the set of reagents or antibiotics described above, then starvation-independent spores are produced. Within a few hours of exposure to any one of these substances, the rod-shaped cells shorten, become spherical, and finally turn bright in a phase-contrast optical system (Fig. (Fig.4).4). Within a few minutes of addition of glycerol, for example, growth ceases, as measured by culture turbidity (4). Such spores are resistant to detergents, sonication, and mild heating (sporulation of wild-type cells carried out in parallel with mutant cells; see all rows in Table Table1).1). Many mutants which cannot form fruiting bodies or starvation-dependent spores due to a developmental defect can still be induced by glycerol, such as the asg, csg, and dev mutants mentioned above. Since the Ω7536 mutant failed to complete the starvation-dependent shape change, the question arose whether glycerol might also elicit an incomplete shape change. As shown in Fig. Fig.4,4, by 3 h after the addition of glycerol, both the wild type and the mutant have responded by changing their shape, although differently. While the wild-type cells become spherical and phase bright, many of the mutant cells become ovoid, not round, and never phase bright. After 24 h of exposure to 0.5 M glycerol, the wild type will have formed many phase-bright spores. These spores tend to stick to each other and to the culture flask (the images for the wild type in Fig. Fig.44 were made from scrapings from the flask surface). While the wild type retained no more than a small percentage of rod-shaped cells at 24 h, the Ω7536 mutant had only rods in the culture. No ring of mutant culture material adhering to the flask was visible.

FIG. 4
Phase-contrast visible-light micrographs of cells exposed to 0.5 M glycerol. (A) The wild-type strain DK1622 (A and B) and the mutant strain DK 7536 (C and D) are shown at 3 h (A and C) and 24 h (B and D). Bar, 10 μm.
TABLE 1
Resistance of glycerol-induced sporulating cells to treatments

Resistance properties.

Table Table11 shows results of tests of the resistance properties of the Ω7536 cells after glycerol induction. Glycerol-induced spore-like cells, rather than fruiting body contents, were tested because they can be obtained in higher yields, in more pure form, and with fewer potential viability-damaging manipulations. Mutant-cell survival is compared in the table to that of similarly treated wild-type cells, with the mutant/wild-type fraction expressed as a percentage. The standard spore assay uses three 10-s cycles of sonication and a 2-h heat treatment at 50°C to distinguish glycerol spores from vegetative cells. No resistance to this treatment was developed by the mutant; nor was there resistance to sonication alone, or to heat alone, or to 0.01% SDS exposure. Compared with the survival of glycerol-treated wild-type cells (taken as 100%), less than 0.01% survival was seen after incubation of the glycerol-treated mutant spores at 40 or 50°C.

Growth in the presence of glycerol.

Closer examination of the mutant revealed that between 9 and 11 h of incubation in CTT plus 0.5 M glycerol, the ovoid cells induced in the first few hours began to elongate, eventually reaching full rod length. This recovery, by the Ω7536 mutant, of sustained growth in the presence of glycerol was confirmed by measurements of the culture turbidity. Upon the addition of glycerol, the turbidity of the mutant culture begins to rise while that of the wild type decreases, never rising above its initial value during the 24-h period of observation (Fig. (Fig.5A).5A). The rise in turbidity in the Ω7536 mutant is continuous after 7 to 8 h of incubation in glycerol, about the time that the spheres, seen under the microscope, were reverting to rods. Since starvation-independent sporulation is induced in a complete medium (CTT plus glycerol), there are ample nutrients for growth. The final turbidity of the mutant culture at 24 h is like that of a saturated wild-type culture. Reversion to the rod shape implies that the spore structure has not been completed in the Ω7536 mutant and that the intermediate cell is able to grow in complete medium, even in the presence of remaining glycerol.

FIG. 5
(A) Cell density after addition of glycerol to a CTT culture. Squares, wild-type (DK1622) culture; circles, a culture of cells carrying the Ω7536 insertion (DK10524). Glycerol was added to the cultures at 0 h, and cell density was measured in ...

Since the mutant does form ovoid cells in response to glycerol, and considering that the change in shape of wild-type cells during fruiting body sporulation correlates with the expression of β-galactosidase from the Ω7536 locus, the question arises whether that correlation would still hold during starvation-independent sporulation. Indeed, β-galactosidase is expressed from the Ω7536 mutant (Fig. (Fig.5B).5B). Expression rises within the 1st h after glycerol addition, compared with 17 h after starvation, in line with the times of shape change in the two induction processes.

Ω7536 and the C-signal.

As shown above, Ω7536 is not expressed in a csgA mutant, which cannot make the C-signal (Fig. (Fig.3).3). In light of this dependence, the substantial induction of β-galactosidase by starvation in the Ω7536 strain implies that it is capable of producing the C-signal. To assess the level of C-factor production of the mutant in vivo, it was cocultured with csgA cells. When a C-signal-deficient mutant, csgA, is codeveloped with wild-type cells, the mutant can be fully rescued (11): it aggregates—the aggregates containing both mutant and wild type cells; it produces a wild-type complement of spores within fruiting bodies; the mutant spores are recognized by their oxytetracycline resistance. Table Table22 shows the results of coculture assays of C-factor production by Ω7536, and they lead to several conclusions. On the one hand, the insertion mutant (unlike a csgA strain) cannot be complemented, or rescued, when mixed with wild-type cells. This indicates that the mutant defect in Ω7536 is cell autonomous relative to C-signaling-induced sporulation. On the other hand, when Ω7536 was mixed with a csgA mutant, the sporulation defect of the csgA mutant was rescued, and the spore levels were wild type. Since spore levels depend on the level of C-factor (12), the Ω7536 mutant thus appears to make wild-type levels of C-signal. Since Ω7536 is dependent on the C-signal for its transcription, Ω7536 is expected to be part of the C-signal response pathway; the question is, which part? Since the Ω7536 mutant can aggregate but cannot complete spore morphogenesis, it would be expected to lie in the sporulation branch of the C-signal response pathway. The dev operon has been identified as a component of that branch (25, 26). The Tn5lac of Ω7536 was transduced into a devRS mutant background, and one of the curves in Fig. Fig.33 shows that Ω7536 is not expressed in the dev mutant background. Thus, Ω7536 depends on dev function, and consequently Ω7536 can be consistently placed just after dev in the sporulation branch of the C-signal response pathway, as represented in Fig. Fig.6.6.

TABLE 2
C-factor assay by rescue of fruiting body sporulation
FIG. 6
C-signal control of sporulation. Ω7536 in the diagram refers to the function normally performed by those genes. The point at which the starvation-independent inducers act is also indicated by an upward-pointing arrow. The C-signal control of FruA ...

DISCUSSION

The starvation-dependent and starvation-independent pathways of sporulation have different time scales, different nutritional requirements, and different inducers. As well, they produce structurally different spores, since fruiting body spores are coated with protein S, which is completely lacking in glycerol spores (13, 28). Nevertheless, the two sporulation pathways share the step indicated by Ω7536 in Fig. Fig.6,6, a step that is necessary to transform a vegetative rod into a spherical spore. The locus identified by insertion Ω7536 is transcribed in response to starvation as well as to glycerol. In fruiting body development, that locus begins to be expressed around 17 h, coordinately with the time of sporulation.

The C-signal triggers first the aggregation and then the sporulation process, as befits fruiting body development (20, 25, 26). These processes are coupled as branches 2 and 3 of Fig. Fig.6.6. As implied by that circuit, the Ω7536 insertion is not expressed during fruiting body development in a mutant that cannot make C-signal (Fig. (Fig.3).3). Moreover, the very severe sporulation defect of Ω7536, decreasing the spore number to 10−6 of the wild-type spore number, is like that of a csgA mutant (Table (Table2).2). The full rescue of csgA mutant sporulation (Table (Table22 shows that this mutant is rescued) implies that the Ω7536 mutant (the rescuer) produces normal levels of C-factor and thus that the positive feedback on csgA expression (branch 1 in Fig. Fig.6)6) is fully active (6). Based on the data of Ellehauge et al. (5), this branch is drawn ahead of FruA because Act1 and not FruA sets the level of C-factor (6). The normal size and shape of Ω7536 aggregates (Fig. (Fig.1)1) implies that branch 2 also functions normally. Expression of β-galactosidase from the lacZ insertion in Ω7536 implies that branch 3 in Fig. Fig.66 is normal up to and including the expression of the Ω7536 locus. Based on these data, uniquely consistent positions can be assigned to the Ω7536 function and to the inducing action of glycerol when it acts as a starvation-independent inducer. These two processes are positioned on the sporulation branch of the C-signaling pathway adjacent to each other. Since Ω7536 expression is blocked in a dev mutant (Fig. (Fig.3),3), and since dev insertion mutations severely reduce sporulation (27), dev must lie upstream of Ω7536 function in the sporulation branch of the pathway. Glycerol induces Ω7536 expression but does not induce dev (Ω4414) expression (16), although dev mutants can be induced to make viable glycerol spores. As demonstrated by Ellehauge et al. (5), dev follows activated FruA, as illustrated by branch 3 of the C-signaling pathway (Fig. (Fig.66).

For the same reasons, the glycerol stimulus that induces sporulation must feed into the C-signal response pathway between dev and the Ω7536 function, which is shown as branch 4 in Fig. Fig.6.6. Since branch 4 enters beyond dev, its position would explain first why glycerol spores lack protein S, which is encoded by tps, because tps has to be induced by A-signaling (18). It would also explain the observation that most developmental mutant defects, including dev and those that precede it, such as asg and csg, do not eliminate glycerol spore inducibility. On the one hand, the nearly 100% induction of sporulation by glycerol implies that every growing cell is equipped to become a spore. On the other hand, fruiting body sporulation, in which only 1% of the input cells become viable spores (Table (Table1),1), may be limited by the residual capacity for protein synthesis in starving cells and by Dev induction of sporulation functions. As yet, not much is known about this induction.

The Ω7536 insertion mutant begins, but is unable to complete, the normally observed change to a stable spherical shape in both fruiting body and glycerol sporulation. Within the mutant aggregates (Fig. (Fig.3)3) are a variety of cellular forms: short rods, bean shapes, and various ovoids—all plausibly intermediates in the normal process of shape change. The virtually continuous spectrum of intermediate forms inside these mutant aggregates suggests a normally continuous transition of shape, instead of a few discrete intermediate shapes. The cellular forms induced by exposing the mutant to glycerol are generally ovoid (Fig. (Fig.4),4), are more sensitive to heat, sonication, and detergents than the wild type (Table (Table1),1), and begin to grow by rod elongation, even in the presence of glycerol. (Nutrients sufficient for rapid growth are present in the glycerol sporulation medium.) The turbidity increase that accompanies the growth of the Ω7536 mutant, after it first becomes ovoid, implies that glycerol has induced only a temporary halt to growth and to cell elongation in the mutant. This temporary growth arrest may open a window on the mechanism of cell shape change in myxosporulation.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant GM23441 from the National Institute of General Medical Sciences to D.K. and by postdoctoral fellowship GM16344 to L.G. from the National Institute of General Medical Sciences.

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